The present experiments represent a logical continuation of our current research efforts to gain a better understanding of the actions and mechanisms of action of nicotine in the mammalian central nervous system. Our strategy is to use a combination of neurochemical, biochemical and electrophysiological approaches to study the actions and mechanism of action of nicotine on dopamine neurons of freely-moving, unanesthetized rats as well as the cellular mechanisms involved in the release of dopamine from culture cell lines. During the present grant period, we examined the effect of nicotine on the firing rate of the cell bodies and the release of DA from the terminals. We observed that nicotine increases the firing rate of A10 neurons to a greater extent than A9, that chronic treatment resulted in a change from stimulation to inhibition of the firing rate, while the release of DA from the terminals was enhanced. The objectives of Specific Aims 1-3 are to examine the mechanisms of these effects of chronic nicotine and to obtain further evidence for a greater sensitivity of A10 over A9 neurons. A combination of electrophysiological and neurochemical studies will be carried out in the freely-moving, unanesthetized rat. Specific Aim 1 will determine the mechanism for the enhancement of the nicotine-induced release of DA from the striatum of freely-moving rats chronically exposed to nicotine. We will measure the number of spontaneously active DA cells, nicotinic-cholinergic receptors as well as DA content, release and synthesis. Specific Aim 2 will examine the nicotine-induced release of DA from terminals of the mesolimbic DA pathway in freely-moving rats acutely and following chronic exposure to nicotine. We will measure the release of newly-synthesized DA using push-pull perfusion and by tissue 3MT. Specific Aim 3 will define the electrophysiological effects of nicotine on mesolimbic DA neurons following chronic treatment with nicotine. In the final specific Aim, we will carry out biochemical studies (DA release, cAMP accumulation, receptor binding) as well as electrophysiological recordings in the PC12 cells, PC12 mutants and primary cultures of bovine chromaffin cells as model systems to come to an understanding of potential intracellular mechanisms involved in the release of DA. Biochemical studies will address the hypothesis that the nicotine-induced release of DA is involved in activation of a calmodulin-sensitive adenylate cyclase leading to an increase in cAMP. These experiments will provide useful new information on the chronic effect and mechanisms of action of nicotine on dopamine neurotransmission in freely-moving, unanesthetized animals and in the intracellular mechanisms involved in the nicotine-induced release of dopamine. They will also further define the importance of the mesolimbic, mesocortical dopamine pathway as the biological substrate for dependence.